Pluripotent stem cell that induces repair and regeneration after myocardial infarction

ABSTRACT

An object of the present invention is to provide a novel medical application for use in regenerative medicine that uses pluripotent stem cells (Muse cells). The present invention provides a cell preparation for treating myocardial infarction, and particularly serious massive myocardial infarction and heart failure associated therewith, that contains pluripotent stem cells positive for SSEA-3 isolated from biological mesenchymal tissue or cultured mesenchymal cells. The cell preparation of the present invention is based on a cardiac tissue regeneration mechanism by which Muse cells are made to selectively accumulate in damaged myocardial tissue and differentiate into cardiac muscle in that tissue as a result of intravenous administration of Muse cells to a subject presenting with the aforementioned disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

The current application is a continuation of U.S. patent applicationSer. No. 14/421,754 filed Feb. 13, 2015, allowed, which is a 371National Phase Application of PCT/JP2013/071981 filed Aug. 15, 2013,which application claims priority to foreign application nos.PCT/JP2013/054049 filed Feb. 19, 2013 and JP 2012-181029 filed Aug. 17,2012, the disclosures of which are hereby incorporated by reference intheir entireties for all purposes.

TECHNICAL FIELD

The present invention relates to a cell preparation used in regenerativemedicine. More particularly, the present invention relates to a cellpreparation containing pluripotent stem cells that are effective forrepairing and regenerating cardiac tissue that has been damaged bymyocardial infarction.

BACKGROUND ART

Myocardial infarction, which is caused by myocardial necrosis broughtabout by coronary artery occlusion, is an important issue to be solvedin clinical medicine since it causes sudden cardiac death and chroniccardiac death. In the case of acute myocardial infarction in particular,the mortality rate is high at 35% to 50%, and 60% to 70% of fatal casesdie within 1 to 2 hours after the attack. In addition, even if patientssurvive the acute stage, in cases in which the myocardial necroticlesion is large following the initial attack, there is a high risk ofsuccumbing to recurrent myocardial infarction or accompanying heartfailure. Thus, in treating myocardial infarction, it is necessary torapidly implement treatment soon after the attack has occurred, and itis important to minimize the size of the necrotized myocardium, namelythe infarct size, as much as possible.

For example, in myocardial infarctions such as severe massive myocardialinfarction, since left ventricular remodeling proceeds leading to heartfailure, prognosis is known to be poor. In general, coronaryrecanalization therapy in the form of thrombolytic therapy orrevascularization is typically performed for myocardial infarction.However, there are many cases in which the effects of recanalization maynot be obtained or conversely, myocardial cells may be damaged byreperfusion injury and the like, and satisfactory therapeutic effectsmay not be obtained by recanalization therapy alone. Consequently,although studies have been conducted on pharmaceutical agents expectedto demonstrate myocardial protective action for use as an adjuvanttherapy to recanalization therapy, a satisfactory pharmaceutical agenthas yet to be found. In addition, in the case of serious massivemyocardial infarction, prognosis would improve if it were possible toregenerate necrotic myocardial tissue and improve left ventricularremodeling. However, there is currently no medical treatment that iseffective against the aforementioned disorders.

As was previously described, in the treatment of myocardial infarction,although it is required to rapidly provide treatment soon after anattack, since there is no definitive treatment method for minimizinginfarct size, efforts are being focused on regenerating the myocardialtissue that has necrotized. In particular, attention has recently beenfocused on biological cells capable of contributing to tissueregeneration. Although known examples of cells obtained from adults thathave the ability to differentiate include mesenchymal stem cells (MSC)that have the ability to differentiate into bone, cartilage, adipocytes,neurons or skeletal muscle and the like (Non-Patent Documents 1 and 2),these consist of cell groups containing various cells, the actual stateof their ability to differentiate is not understood, and there have beenconsiderable fluctuations in therapeutic effects. In addition, althoughiPS cells (Patent Document 1) have been reported to be adult-derivedpluripotent stem cells, in addition to the establishment of iPS cellsrequiring an extremely complex procedure involving the introduction ofspecific genes into mesenchymal cells in the form of a skin fibroblastfraction and the introduction of specific compounds into somatic cells,since iPS cells have a high tumorigenic potential, extremely highhurdles must be overcome for their clinical application.

It has been determined from research by M. Dezawa, one of the inventorsof the present invention, that multilineage-differentiating stressenduring cells (Muse cells) expressing surface antigen in the form ofstage-specific embryonic antigen-3 (SSEA-3), which are present inmesenchymal cell fractions and can be obtained without going through aninduction procedure, are responsible for the pluripotency possessed bymesenchymal cell fractions, and that they have the potential forapplication to disease treatment aimed at tissue regeneration. Inaddition, Muse cells were also determined to be able to be concentratedby stimulating mesenchymal cell fractions with various types of stress(Patent Document 2, Patent Document 3). However, there have yet to beany examples of the use of Muse cells for the prevention and/ortreatment of myocardial infarction such as serious massive myocardialinfarction or its accompanying heart failure, and the obtaining ofanticipated therapeutic effects has yet to be clearly determined.

PRIOR ART DOCUMENT Patent Documents

-   [Patent Document 1] Japanese Patent No. 4183742-   [Patent Document 2] International Publication No. WO 2011/007900

Non-Patent Documents

-   [Non-Patent Document 1] Dezawa, M., et al., J. Clin. Invest., Vol.    113, p. 1701-1710 (2004)-   [Non-Patent Document 2] Dezawa, M., et al., Science, Vol. 309, p.    314-317 (2005)-   [Non-Patent Document 3] Wakao, S., et al., Proc. Natl. Acad. Sci.    USA, Vol. 108, p. 9875-9880 (2011)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a novel therapeuticapplication using pluripotent stem cells (Muse cells) in regenerativemedicine. More specifically, an object of the present invention is toprovide a cell preparation for the treatment of myocardial infarction(and particularly, serious massive myocardial infarction), as well asthe prevention and/or treatment of its accompanying heart failure, thatcontains Muse cells.

Means for Solving the Problems

The inventors of the present invention found that, by administering Musecells by intravenous injection for myocardial infarction induced bycoronary artery ischemia (30 minutes) using Japanese white rabbits, theMuse cells accumulate locally in damaged myocardial tissue,differentiate into myocardial cells within the damaged myocardialtissue, and bring about a reduction in infarct size and improvement orrestoration of cardiac function, thereby leading to completion of thepresent invention.

Namely, the present invention is as described below.

[1] A cell preparation for treating myocardial infarction, containingpluripotent stem cells positive for SSEA-3 isolated from biologicalmesenchymal tissue or cultured mesenchymal cells.

[2] The cell preparation described in [1], wherein the pluripotent stemcells positive for SSEA-3 contain a concentrated cell fraction as aresult of stimulation by external stress.

[3] The cell preparation described in [1] or [2] above for preventionand/or treatment of heart failure following serious massive myocardialinfarction in humans.

[4] The cell preparation described in [1] to [3] above, wherein thepluripotent stem cells are CD105-positive.

[5] The cell preparation described in [1] to [4] above, wherein thepluripotent stem cells are CD117-negative and CD146-negative.

[6] The cell preparation described in [1] to [5] above, wherein thepluripotent stem cells are CD117-negative, CD146-negative, NG2-negative,CD34-negative, vWF-negative and CD271-negative.

[7] The cell preparation described in [1] to [6] above, wherein thepluripotent stem cells are CD34-negative, CD117-negative,CD146-negative, CD271-negative, NG2-negative, vWF-negative,Sox10-negative, Snail-negative, Slug-negative, Tyrp1-negative andDct-negative.

[8] The cell preparation described in [1] to [7], wherein thepluripotent stem cells are pluripotent stem cells having all of theproperties indicated below:

(i) low or absent telomerase activity;

(ii) ability to differentiate into any of the three germ layers;

(iii) absence of demonstration of neoplastic proliferation; and,

(iv) self-renewal ability.

[9] The cell preparation described in [1] to [8] above, wherein thepluripotent stem cells have the ability to accumulate at the site ofmyocardial infarction.

[10] The cell preparation described in [1] to [9] above, wherein thepluripotent stem cells have the ability to differentiate into vascularendothelial cells.

[11] The cell preparation described in [1] to [9] above, wherein thepluripotent stem cells have the ability to differentiate into myocardialcells.

[12] The cell preparation described in [1] to [11] above, wherein thepluripotent stem cells are administered into a vein or coronary arteryof a subject within 1 month after ischemia one to ten times in atherapeutically effective amount of 1×10³ cells/individual to 1×10⁶cells/individual.

[13] The cell preparation described in [1] to [12] above, wherein thesize of the myocardial infarct is reduced in comparison with anon-administered control.

[14] The cell preparation described in [1] to [13] above, wherein atleast one cardiac function indicator, selected from the group consistingof change in left ventricular pressure over time, left ventricularend-diastolic dimension (LVDd), ejection fraction (EF), left ventricularfractional shortening (FS) and left ventricular end-systolic dimension(LVDs), is restored to the normal value.

Effects of the Invention

The present invention is able to dramatically reduce the size of amyocardial infarct by means of a cardiac tissue regeneration mechanismby which Muse cells are made to selectively accumulate in damagedmyocardial tissue and differentiate into cardiac muscle following theiradministration into a vein or the like of a subject suffering frommyocardial infarction, and particularly serious massive myocardialinfarction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts sections of cardiac tissue containing an infarcted siteobtained from a rabbit model of myocardial infarction. The infarctedsites were determined by triphenyl tetrazolium chloride (TTC) staining.The non-stained regions surrounded by broken lines indicate theinfarcted sites. The right panel is a tissue section obtained 14 daysafter reperfusion that was reperfused 30 minutes after ischemia followedby intravenous administration of Muse cells 24 hours after reperfusion.The left panel is a tissue section in which physiological saline wasadministered intravenously instead of Muse cells. Transplantation ofMuse cells resulted in a significant decrease in the size of theinfarcted site.

FIG. 2 is a bar graph depicting infarct size calculated as the ratio (%)of infarcted regions to ischemic regions following determination ofinfarcted sites by TTC staining. The left bar indicates the averagevalue of infarct size for a physiological saline control group (n=3),while the right bar indicates the average value of infarct size in aMuse cell transplant group (n=4). Transplantation of Muse cells resultedin a significant decrease in infarct size.

FIG. 3 shows the results of a histological examination of reductioneffects on infarct size as determined by Masson's trichrome (MT)staining. In MT staining, tissue composed of viable cells is stainedred, while tissue of infarcted regions exhibiting collagen fibrosis isnot stained and appears pale. A Muse cell transplant group (n=4)demonstrated a smaller infarcted region and a considerable reduction ininfarct size in comparison with a control group administeredphysiological saline (n=3). In addition, although reduction of infarctsize was observed in tissue transplanted with a mesenchymal stem cell(MSC) fraction, reduction effects on infarct size were less potent incomparison with the Muse cell transplant group.

FIG. 4 depicts differentiation of Muse cells into myocardial cells incardiac tissue. Infarcted sites and non-infarcted sites aredistinguished with the broken line shown as a boundary line in the leftpanel. Myocardial cells were stained red by rhodamine-phalloidinstaining (center panel). In addition, Muse cells preliminarilyintroduced with green fluorescent protein (GFP) gene prior to transplantusing a lentivirus can be seen to be localized at infarcted sites (rightpanel). The result of superimposing these two images is shown in theleft panel. As a result, a large number of cells where red and greencolors overlap are present at the infarcted sites, thereby suggestingthat transplanted Muse cells differentiate into myocardial cells.

FIG. 5 shows the results of examining the differentiated state of Musecells that have integrated into infarcted sites. The presence or absenceof expression of atrial natriuretic peptide (ANP), which is known to beexpressed in juvenile myocardial cells, was examined by fluorescentstaining. The green color indicates GFP stain (Muse cells), the redcolor indicates ANP stain and the blue color indicates DAPI stain (whichis used to stain cell nuclei). In the left panel, these three types offluorescence are observed within a plurality of the same cells, therebysuggesting that cells are included among the transplanted Muse cellsthat are differentiating into myocardial cells.

FIG. 6 indicates the results of examining cardiac function followingMuse cell transplant using changes in blood pressure over time as anindicator (±dp/dt, p: blood pressure, t: time). The upper panelindicates the results of measuring ventricular systolic function(+dp/dt), while the lower panel indicates the results of measuringventricular diastolic function (−dp/dt). These results suggest thatcardiac function in a group of rabbits transplanted with Muse cellsimproved significantly in comparison with a control group.

FIG. 7 depicts 2D echocardiograms of parasternal long axiscross-sections of the left ventricle. The left panel indicates an imageof the left ventricle of a rabbit administered physiological saline(control), while the right panel indicates an image of the leftventricle of a rabbit transplanted with Muse cells. Indicators ofcardiac function consisting of LVDd (left ventricular end-diastolicdimension), ejection fraction (EF) and left ventricular fractionalshortening (FS) were measured based on these images. Each of thesevalues was shown to be restored to normal in the Muse cell transplantgroup.

FIG. 8 is a graph depicting infarct size calculated as the ratio (%) ofthe infarcted region to the left ventricle following determination ofinfarcted sites by Masson's trichrome staining. Infarct size (whitecircles) and the average values thereof (black dots) of a physiologicalsaline control group (n=10), an MSC (mesenchymal stem cell fraction)transplant group (n=10), a Muse cell transplant group (n=10), and anon-Muse cell group (MSC cells not including Muse cells) (n=4) arerespectively plotted on the horizontal axis moving from left to right.Transplantation of Muse cells resulted in a significant reduction ininfarct size in the same manner as in FIG. 2.

FIG. 9 shows the results of a histological examination of reductioneffects on infarct size as determined by Masson's trichrome (MT)staining in the same manner as FIG. 3. FIG. 9 depicts photographs of onesample randomly selected from each transplant group corresponding to theresults shown in FIG. 8.

FIG. 10 shows the results of examining the differentiated state of Musecells that have integrated into infarcted sites. The presence or absenceof expression of troponin I, which is known to be a myocardial marker,was examined by fluorescent staining in the same manner as FIG. 4. Thegreen color indicates GFP stain (Muse cells), the red color indicatestroponin I stain and the blue color indicates DAPI stain (which is usedto stain cell nuclei). In the left panel, these three types offluorescence are observed within a plurality of the same cells, therebysuggesting that cells are included among the transplanted Muse cellsthat are differentiating into myocardial cells.

FIG. 11 indicates the results of examining cardiac function followingMuse cell transplant using changes in blood pressure over time as anindicator (±dp/dt, p: blood pressure, t: time) in the same manner asFIG. 6. +dp/dt indicates systolic function while −dp/dt indicatesdiastolic function. These results suggest that cardiac function in agroup of rabbits transplanted with Muse cells improved significantly incomparison with a control group, MSC transplant group and non-Muse celltransplant group.

FIG. 12 indicates the results of measuring indicators of cardiacfunction consisting of LVDd (left ventricular end-diastolic dimension),left ventricular end-systolic dimension (LVDs), ejection fraction (EF)and left ventricular fractional shortening (FS) from 2D echocardiograms.Each of these values was shown to be restored to normal in the Muse celltransplant group.

FIG. 13 shows the results of examining the differentiated state of Musecells that have integrated into infarcted sites. The presence or absenceof expression of CD31, which is known to be a vascular endothelial cellmarker, was examined. Since CD31-positive microvascular density was highat infarcted sites of tissue transplanted with Muse cells in comparisonwith other transplant groups, the possibility is suggested that thetransplant Muse cells differentiate into vascular endothelial cells.Microvascular density as observed in a high power field (HPF) is plottedon the vertical axis.

MODE FOR CARRYING OUT THE INVENTION

The present invention relates to a cell preparation for treatingmyocardial infarction that contains SSEA-3-positive pluripotent stemcells (Muse cells). The following provides a detailed explanation of thepresent invention.

1. Applicable Diseases

The present invention is used for the purpose of treating myocardialinfarction, and particularly serious massive myocardial infarction,along with its accompanying heart failure, using a cell preparationcontaining SSEA-3-positive pluripotent stem cells (Muse cells). Here,“myocardial infarction” refers to myocardial necrosis brought about bycoronary artery occlusion. In addition, “heart failure” refers to asyndrome caused by the failure of cardiac function to circulate anadequate amount of blood, and includes a decrease in cardiac output andan accompanying increase in venous pressure as well as various clinicalsymptoms occurring as a result thereof. Myocardial infarction is a causeof sudden cardiac death and chronic cardiac death. In the case of acutemyocardial infarction in particular, the mortality rate is high at 35%to 50%, and 60% to 70% of fatal cases die within 1 to 2 hours after theattack. In addition, even if patients survive the acute stage, in casesin which the myocardial necrotic lesion is large following the initialattack, there is a high risk of succumbing to recurrent myocardialinfarction. Thus, in treating myocardial infarction, it is necessary torapidly implement treatment soon after the attack has occurred, and itis important to minimize the size of the necrotized myocardium, namelythe infarct size, as much as possible. In addition, variousclassifications are used to assess the severity of myocardialinfarction. Examples thereof include classification according to theamount of elapsed time, morphological classification (range within themyocardium, site, and/or size of necrosis, etc.), form of myocardialnecrosis, post-infarction ventricular remodeling, hemodynamicclassification (as related to treatment, prevention and the like) andclassification according to clinical severity. Here, myocardialinfarction having a high degree of severity in which myocardial necrosiscovers a wide range in particular is referred to as “severe massivemyocardial infarction”. An example thereof is complete occlusion of thedistal portion of the left coronary artery. In addition, severe massivemyocardial infarction is known to be associated with a poor prognosissince left ventricular remodeling of cardiac muscle proceeds resultingin heart failure. Here, “left ventricular remodeling” followingmyocardial infarction refers to a series of changes, includinghypertrophy of myocardial cells, increased interstitium (extracellularmatrix) and enlargement of the cardiac lumen, that occur compensatory todecreased cardiac function caused by thinning of the infarcted site thatoccurs following myocardial infarction. Since long-term prognosisfollowing myocardial infarction is correlated with the degree of leftventricular dysfunction, inhibition of left ventricular remodeling isessential for maintaining and preserving the function of the leftventricle.

In general, in the case of myocardial infarction within 6 hours ofattack, aggressive reperfusion therapy of the occluded coronary arterymakes it possible to reduce the necrotized range of cardiac muscle. Inaddition to this therapy, it is meaningful to perform reperfusiontherapy in cases in which 24 hours or less have elapsed since attack. Inthe case of the acute stage, coronary artery disease is frequentlytreated using a catheter. In contrast, the cell preparation of thepresent invention can be targeted for treatment of cases in which theamount of time until reperfusion is extremely long or cases in whichreperfusion and catheterization were ineffective. In other words,according to the present invention, a cell preparation containing Musecells is provided for the purpose of treating myocardial infarctionbased on regeneration of cardiac tissue, including prevention of theoccurrence of heart failure caused by left ventricular remodeling.

2. Cell Preparation

(1) Pluripotent Stem Cells (Muse Cells)

The existence of the pluripotent stem cells used in the cell preparationof the present invention in the body was discovered by M. Dezawa, one ofthe applicants of the present invention, and the cells were named“multilineage-differentiating stress enduring (Muse) cells”. Muse cellscan be obtained from bone marrow aspirates or skin tissue such as dermalconnective tissue, and are sporadically present in the connective tissueof various organs. In addition, these cells have both the properties ofpluripotent stem cells and mesenchymal stem cells, and are identified asbeing double-positive for each of the cell surface markers of“stage-specific embryonic antigen-3 (SSEA-3)” and “CD105”. Thus, Musecells or cell populations containing Muse cells can be isolated frombody tissue by using these antigen markers as indicators. In addition,since Muse cells are resistant to stress, they can be concentrated frombiological mesenchymal tissue or cultured mesenchymal cells bystimulating with various types of stress. A cell fraction in which Musecells have been concentrated by stress stimulation can also be used forthe cell preparation of the present invention. Details regarding methodsused to isolate, identify and concentrate Muse cells as well as theircharacteristics are disclosed in International Publication No. WO2011/007900. In addition, as has been reported by Wakao, et al. (2011,previously cited), in the case of using a cell culture obtained byculturing mesenchymal cells present in bone marrow, skin and the like asthe parent population of Muse cells, all cells positive for SSEA-3 areknown to be positive for CD105. Thus, in the cell preparation of thepresent invention, in the case of isolating Muse cells from biologicalmesenchymal tissue or cultured mesenchymal cells, Muse cells can bepurified and used simply by using SSEA-3 as an antigen marker.Furthermore, in the present description, pluripotent stem cells (Musecells) able to be used in a cell preparation for treating myocardialinfarction that have been isolated from biological mesenchymal tissue orcultured mesenchymal cells by using SSEA-3 as an antigen marker, or acell population containing Muse cells, may simply be described as“SSEA-3-positive cells”.

Simply speaking, Muse cells or cell populations containing Muse cellscan be isolated from biological tissue (such as mesenchymal tissue)using antibody to the cell surface marker SSEA-3 alone or using antibodyto SSEA-3 and CD105, respectively. Here, “biological tissue” refers tothe biological tissue of a mammal. In the present invention, although anembryo in a development stage prior to a fertilized egg or blastulastage is not included in biological tissue, an embryo in a developmentstage in or after the fetus or blastula stage, including the blastula,is included. Examples of mammals include, but are not limited to,primates such as humans or monkeys, rodents such as mice, rats, rabbitsor guinea pigs as well as cats, dogs, sheep, pigs, cows, horses,donkeys, goats and ferrets. The Muse cells used in the cell preparationof the present invention are clearly distinguished from embryonic stem(ES) cells and embryonic germ (EG) cells in that they are derived frombiological tissue. In addition, “mesenchymal tissue” refers to tissue ofbone, cartilage, fat, blood, bone marrow, skeletal muscle, dermis,ligaments, tendons or heart and the like, as well as connective tissuethereof. For example, Muse cells can be obtained from bone marrow andskin. In addition, an object of the present invention is to provide acell preparation used for the purpose of regenerating cardiac muscle,and for example, Muse cells are preferably used that have been isolatedfrom mesenchymal tissue collected from the living body. In addition,Muse cells may also be isolated from cultured mesenchymal cells usingthe aforementioned isolation means. Furthermore, Muse cells used in thecell preparation of the present invention may be autologous or allogenicrelative to the recipient who receives the cell transplant.

As has been described above, although Muse cells or cell populationscontaining Muse cells can be isolated from biological tissue by usingtheir property of being SSEA-3-positive and CD105-positive, human adultskin is known to contain various types of stem cells and precursorcells. However, Muse cells are not the same as these cells. Examples ofsuch stem cells and precursor cells include skin-derived precursor (SKP)cells, neural crest stem cells (NCSC), melanoblasts (MB), perivascularcells (PC), endothelial precursor (EP) cells and adipose-derived stemcells (ADSC). Muse cells can be isolated from these cells by using“non-expression” of a unique marker as an indicator of these cells. Morespecifically, Muse cells can be isolated by using non-expression of atleast one of 11 markers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11markers, selected from the group consisting of CD34 (marker for EP andADSC), CD117 (c-kit) (MB marker), CD146 (PC and ADSC marker), CD271(NGFR) (NCSC marker), NG2 (PC marker), vWF factor (von Willebrandfactor) (EP marker), Sox10 (NCSC marker), Snail (SKP marker), Slug (SKPmarker), Tyrp1 (MB marker) and Dct (MB marker). For example, althoughnot limited thereto, Muse cells can be isolated by using non-expressionof CD117 and CD146 as an indicator, can be isolated using non-expressionof CD117, CD146, NG2, CD34, vWF and CD271 as an indicator, and can beisolated by using non-expression of the aforementioned 11 markers as anindicator.

In addition, Muse cells having the aforementioned characteristics usedin the cell preparation of the present invention may have at least oneproperty selected from the group consisting of:

(i) low or absent telomerase activity;

(ii) ability to differentiate into any of the three germ layers;

(iii) absence of demonstration of neoplastic proliferation; and,

(iv) self-renewal ability.

In one aspect of the present invention, the Muse cells used in the cellpreparation of the present invention have all of the aforementionedproperties. Here, with respect to the aforementioned (i), “low or absenttelomerase activity” refers to telomerase activity being low or beingunable to be detected in the case of having detected telomerase activityusing, for example, the Trapeze XL Telomerase Detection Kit (MilliporeCorp.). “Low” telomerase activity refers to having telomerase activityroughly equal to that of human fibroblasts, for example, or havingtelomerase activity that is ⅕ or less and preferably 1/10 or less incomparison with Hela cells. With respect to the aforementioned (ii),Muse cells have the ability to differentiate into the three germ layers(endoderm, mesoderm and ectoderm) in vitro and in vivo, and by inducingto differentiate by culturing in vitro, for example, can differentiateinto skin, liver, nerve, muscle, bone or fat and the like. In addition,Muse cells may also demonstrate the ability to differentiate into thethree germ layers in the case of transplanting in vivo into testes, forexample. Moreover, Muse cells also have the ability to migrate, graftand differentiate into a damaged organ (such as the heart, skin, spinalcord, liver or muscle) by being transplanted into the body byintravenous injection. With respect to the aforementioned (iii),although Muse cells proliferate at a growth rate of about 1.3 days in asuspension culture, they also have the property of discontinuingproliferation for about 10 days, and in the case of having beentransplanted into testes, have the property of not becoming malignantfor at least six months. In addition, with respect to the aforementioned(iv), Muse cells have self-renewal (self-replication) ability. Here,“self-renewal” refers to culturing cells contained in an embryoidbody-like cell mass obtained by suspension culturing single Muse celland allowing them to reform an embryoid body-like cell mass.Self-renewal may be carried out for one cycle or repeated for aplurality of cycles.

In addition, a cell fraction containing Muse cells used in the cellpreparation of the present invention may be a cell fraction obtained bya method consisting of applying an external stress stimulus tobiological mesenchymal tissue or cultured mesenchymal cells, eradicatingthose cells other than cells that are resistant to the external stress,and recovering the surviving cells, in which SSEA-3-positive andCD105-positive pluripotent stem cells having all of the propertiesindicated below have been concentrated:

(i) SSEA-3 positive;

(ii) CD105-positive;

(iii) low or absent telomerase activity;

(iv) ability to differentiate into three germ layers;

(v) absence of demonstration of neoplastic proliferation; and,

(vi) self-renewal ability.

The aforementioned external stress may consist of any of proteasetreatment, culturing at a low oxygen concentration, culturing underphosphate-deficient conditions, culturing under serum-deficientconditions, culturing under poor nutritional conditions, culturing underexposure to heat shock, culturing under mechanical stimulation,culturing under shaking treatment, culturing under pressure treatmentand physical shock or a combination of a plurality thereof.

The duration of the aforementioned protease treatment in order to impartexternal stress to cells is preferably a total of 0.5 hours to 36 hours.In addition, the protease concentration is the concentration used whenexfoliating cells that have adhered to the culture vessel, when breakingup a cell mass into single cells, or when recovering single cells fromtissue.

The aforementioned protease is preferably a serine protease, asparticacid protease, cysteine protease, metalloprotease, glutamic acidprotease or N-terminal threonine protease. The aforementioned proteaseis more preferably trypsin, collagenase or dispase.

In addition, Muse cells having the aforementioned characteristics usedin the cell preparation of the present invention accumulate in damagedmyocardial tissue (site of myocardial infarction) following intravenousadministration as will be subsequently described, and as a result ofdifferentiating into myocardial cells in that tissue, are able to reduceinfarct size and enable cardiac function to improve or return to normal(Examples 2 to 4).

(2) Preparation and Use of Cell Preparation

The cell preparation of the present invention, although not limitedthereto, is obtained by suspending Muse cells or a cell populationcontaining Muse cells obtained in the aforementioned (1) inphysiological saline or a suitable buffer (such as phosphate-bufferedphysiological saline). In this case, in the case the number of Musecells isolated from autologous or allogenic tissue is low, cells may becultured prior to cell transplant and allowed to proliferate until aprescribed cell concentration is obtained. Furthermore, as has beenpreviously reported (International Publication No. WO 2011/007900),since Muse cells do not undergo neoplastic transformation, there islittle likelihood of the cells becoming malignant even if cellsrecovered from biological tissue are contained that have still notdifferentiated, thereby making them safe. In addition, although thereare no particular limitations thereon, culturing of recovered Muse cellscan be carried out in an ordinary growth medium (such as minimumessential medium-α (α-MEM) containing 10% bovine calf serum). Morespecifically, a solution containing a prescribed concentration of Musecells can be prepared by selecting media, additives (such as antibioticsand serum) and the like suitable for the culturing and proliferation ofMuse cells with reference to the aforementioned InternationalPublication No. WO 2011/007900. In the case of administering the cellpreparation of the present invention for treatment of myocardialinfarction to a human, roughly several milliliters of bone marrowaspirate are collected from human ilium, and after isolating Muse cellsby using an antigen marker for SSEA-3 as an indicator, the cells areallowed to proliferate by culturing for an appropriate amount of timeuntil an effective therapeutic dose is reached (such as for 2 to 3weeks), followed by preparing autologous Muse cells in the form of acell preparation.

In addition, when using the cell preparation of Muse cells,dimethylsulfoxide (DMSO) or serum albumin for protecting the cells, orantibiotics and the like for preventing contamination and growth ofbacteria, may also be contained in the cell preparation. Moreover, otherpharmaceutically allowable components (such as a carrier, vehicle,disintegrating agent, buffer, emulsifier, suspending agent, soothingagent, stabilizer, storage agent, preservative or physiological saline),or cells or components other than Muse cells contained in mesenchymalcells, may also be contained in the cell preparation. A person withordinary skill in the art is able to add these factors andpharmaceutical agents to a cell preparation at suitable concentrations.In this manner, Muse cells can be used in the form of a pharmaceuticalcomposition containing various types of additives.

The number of Muse cells contained in the cell preparation prepared inthe manner described above can be suitably adjusted in consideration ofthe gender, age and body weight of the subject, disease state and statein which the cells are used so as to obtain the desired effect intreatment of myocardial infarction (such as reduction of infarct size orimprovement of cardiac function). In Examples 1 to 4 to be subsequentlydescribed, although a rabbit model of myocardial infarction was producedand various types of effects of transplanting Muse cells were examined,extremely superior effects were obtained by administering SSEA3-positivecells to Japanese white rabbits weighing about 2 kg to 3 kg at 5×10⁵cells/animal. On the basis of this result, superior effects can beexpected to be obtained by administering 1.7×10⁵ to 2.5×10⁵ cells/kg perindividual mammal based on body weight. On the other hand,SSEA-3-positive cells may be contained in a cell preparation at 1×10⁶cells/individual or less, for example, as the amount per singleadministration in order to prevent vascular occlusion attributable toadministration of cells. Here, examples of individuals include, but arenot limited to, rabbits and humans. In addition, the cell preparation ofthe present invention may be administered a plurality of times (such as2 to 10 times) at a suitable interval (such as twice per day, once perday, twice per week, once per week or once every two weeks) until thedesired therapeutic effect is obtained. Thus, although dependent uponthe status of the subject, the therapeutically effective dose ispreferably administered, for example, 1 to 10 times at 1×10³ cells to1×10⁶ cells per individual. Although there are no particular limitationsthereon, examples of total individual doses include 1×10³ cells to 1×10⁷cells, 1×10⁴ cells to 5×10⁶ cells, 2×10⁴ cells to 2×10⁶ cells and 5×10⁴cells to 1×10⁶ cells.

The Muse cells used in the cell preparation of the present inventionhave the property of accumulating at a site of myocardial infarction.Thus, in administering the cell preparation, there are no limitations onthe administration site or type of vessel to which the cell preparationis administered (veins and arteries). Examples of veins suitable foradministration include, but are not limited to, the ear vein and jugularvein. In the case of a human, the cubital vein is preferable. Inaddition, examples of arteries suitable for administration include, butare not limited to, a coronary artery. However, in consideration of suchfactors as cell transport efficiency and rapid recovery of the subjectfollowing surgery, the cell preparation is preferably administereddirectly into a coronary artery at an infarcted site by transdermalinsertion of a cardiac catheter. Although there are no limitations onthe puncture site of the cardiac catheter, examples thereof include thewrist (radial artery), elbow (brachial artery) and groin (femoralartery).

Although the cell preparation of the present invention is targeted forthe treatment of serious massive myocardial infarction and other typesof myocardial infarction, the time of administration is presumed to be arange extending from several hours to several weeks following ischemia.Thus, although there are no limitations thereon, the time ofadministration of the cell preparation of the present invention ispreferably no later than within one month following ischemia. The timeof administration is more preferably within 14 days, even morepreferably within 7 days, still more preferably within 72 hours, evenmore preferably within 48 hours, even more preferably within 24 hours,still more preferably within 12 hours and most preferably within 6 hoursfollowing ischemia. Since the target of treatment using the cellpreparation according to the present invention consists of cases inwhich the amount of time until reperfusion is extremely long or cases inwhich reperfusion and catheterization have been ineffective, it isextremely useful for treating myocardial infarction. In addition, sincethe Muse cells used have been confirmed in an experiment conducted bythe inventors of the present invention to not induce an immune reactioneven in the case of being derived from a allogenic source, there are nolimitations on the number of administrations thereof, and may besuitably administered until the desired effect of treatment ofmyocardial infarction is obtained.

In an embodiment of the present invention, infarct size in a subjectpresenting with myocardial infarction can be reduced by administeringthe cell preparation of the present invention. Here, “infarct size” whenused in the present description is defined as the ratio (%) of aninfarcted region to an ischemic region. Here, an ischemic region isdetermined using Evan's blue staining, and non-ischemic regions arestained by this stain. On the other hand, an infarcted region isdetermined by triphenyl tetrazolium chloride (TTC) staining. Moreover,in the case of examining the effect of the cell preparation of thepresent invention on reducing infarct size, it is useful to use thereduction rate relative to the infarct size of a control (namely,(infarct size of control−infarct size following cell transplant)/infarctsize of control×100). According to the present invention, infarct sizeis preferably reduced to 100% relative to a group not administered thecell preparation (control). Infarct size is more preferably reduced to10% to 90%, even more preferably to 20% to 70%, and still morepreferably to 30% to 50%. Furthermore, as indicated in Example 2 to besubsequently described, in the case of using a rabbit model ofmyocardial infarction, in contrast to infarct size having been anaverage of 30.4% in a control group, infarct size in a Muse celltransplant group was an average of 18.2%. On the basis of these values,infarct size can be understood to have been reduced by(30.4−18.2)/30.4×100=about 40% as a result of transplanting Muse cells.

In an embodiment of the present invention, the cell preparation of thepresent embodiment is able to improve cardiac function followingmyocardial infarction or restore to normal (or normal values).Improvement of cardiac function when used in the present descriptionrefers to alleviation and inhibition of the progression of varioussymptoms associated with myocardial infarction, and preferably refers toalleviation of symptoms to a degree that there is no impairment of dailylife. In addition, restoring cardiac function to normal refers to allsymptoms attributable to myocardial infarction returning to the stateprior to myocardial infarction. Furthermore, in one mode of the presentinvention, the cell preparation of the present invention can be used toprevent and/or treat (chronic) heart failure following myocardialinfarction.

Here, typical examples of indicators used to evaluate myocardialfunction include, but are not limited to, changes in blood pressure ofthe left ventricle over time (±dp/dt, p: blood pressure, t: time), leftventricular end-diastolic dimension (LVDd), ejection fraction (EF), leftventricular fractional shortening (FS) and left ventricular end-systolicdimension (LVDs). Improvement or restoration of cardiac function by thecell preparation of the present invention can be assessed using at leastone of the aforementioned five indicators. For example, as described inExample 4, with respect to changes in blood pressure of the leftventricle over time (±dp/dt), although +dp/dt represents cardiacsystolic function while −dp/dt represents cardiac diastolic function ofthe left ventricle, cardiac function was determined to be significantlyimproved in a cell transplant group in comparison with a control groupbased on both measured values (FIGS. 6 and 11). Moreover, with respectto LVDd, although an increase in LVDd was observed in a control groupbased on the results of 2D echocardiography, an increase in LVDd was notobserved in a Muse cell transplant group and was within the normal range(FIGS. 7 and 12). Next, although ejection fraction (EF), which is one ofthe indicators of cardiac systolic function, is considered to be normalat a value of 55% or higher, since EF values were 60.9% (FIG. 7) and anaverage of 59.3% (n=10) (FIG. 12) in a Muse cell transplant group usinga rabbit model of myocardial infarction, EF values were suggested tohave returned to normal. In addition, with respect to left ventricularfractional shortening (FS) as well, which is also an indicator ofcardiac systolic function in the same manner as EF, FS values were 30.4%(FIG. 7) and an average of 30.0% (n=10) (FIG. 12) in rabbitstransplanted with Muse cells. Since the normal value for FS in humans isconsidered to be between 30% and 50%, FS values were suggested to havereturned to normal with respect to this rabbit model of myocardialinfarction as well.

Although the following provides a more detailed explanation of thepresent invention through examples thereof, the present invention is notlimited in any way by these examples.

EXAMPLES Example 1: Production of Rabbit Model of Myocardial Infarction

The protocol for experimentation using rabbits in the present examplewas approved by the ethics committee regarding animal experimentation ofGifu University, and was carried out in line with “Guidelines for theCare and Use of Laboratory Animals” (1996 revised edition) issued by theU.S. National Institutes of Health (NIH). More specifically, theprocedure was as described below. First, Japanese white rabbits (bodyweight: approx. 2 to 3 kg/animal) were anesthetized using sodiumpentobarbital at 30 mg/kg. The rabbits were continuously subjected toanalysis of arterial blood gas, and ventilation conditions were suitablyadjusted so that arterial blood gas was maintained within thephysiological range. The left carotid artery and jugular vein werecannulated followed by monitoring of arterial pressure. After performingleft thoracotomy at the third intercostal space, the heart was exposedand the center of the outer anterior surface of the left ventricle wasligated with 4-0 silk thread below a branch of the descending aorta. Anarrow vinyl tube was passed over both ends of the suture thread, andthe branch of the aorta was occluded by pulling on this suture thread.Next, the tube was fixed in position by clamping using mosquitohemostatic forceps. Myocardial ischemia was confirmed by the presence oflocal cyanosis and electrocardiogram changes. The duration of occlusion(ischemia) was suitably adjusted. After releasing the suture thread,cardiac muscle was confirmed to change to a red color throughout theentire critical area (refer to Yasuda, et al., Am. J. Physiol. HeartCirc. Physiol., 296, p. 1558-1565, 2009).

Example 2: Reduction Effect on Myocardial Infarct Size ofTransplantation of Muse Cells

(1) Preparation of Muse Cells

Bone marrow cells were collected from rabbits (body weight: approx. 2 kgto 3 kg/animal) and SSEA-3-positive cells (Muse cells) were isolatedusing FACS. More specifically, Muse cells were isolated in compliancewith the method described in International Publication No. WO2011/007900 relating to isolation and identification of human Musecells. Furthermore, the Muse cells used for transplant were derived frombone marrow cells of rabbit individuals in which myocardial infarctionhad been induced, adhesive mesenchymal cells were cultured from bonemarrow, and lentivirus-GFP was introduced into the cells after allowingthe cells to proliferate. Muse cells or a cell population containingMuse cells labeled with GFP were isolated with FACS to obtain cellsdouble-positive for GFP and SSEA-3. Subsequently, the cells wereadjusted to a prescribed concentration and returned to the samemyocardial infarcted rabbit by intravenous injection. Furthermore, aswas previously described, in the case of using cells obtained byculturing mesenchymal cells such as bone marrow cells as a parentpopulation of Muse cells, all SSEA-3-positive cells are known to bepositive for CD105 as reported by Wakao, et al. (2001, previouslycited).

(2) Reduction Effect on Myocardial Infarct Size of Transplantation ofMuse Cells

The duration of infarction (ischemia) induced by ligation in the rabbitswas made to be 30 minutes (corresponding to a duration of ischemia inhumans of 3 hours), followed by initiating reperfusion by releasing thesuture thread. SSEA-3-positive cells (5×10⁵ cells), for whichconcentration was adjusted with physiological saline after having beenobtained as described in (1) above, were administered into an ear veinof the rabbits. In addition, a mesenchymal cell fraction (MSC) (5×10⁵cells) and physiological saline were respectively administered intodifferent rabbits for use as comparative controls followed byreperfusion. A comparative study of the reducing effect on infarct sizeof the Muse cells was then carried out 14 days after reperfusion.

More specifically, the rabbits were sacrificed 14 days after reperfusionby treating with heparin (500 U/kg) and intravenously injecting anexcess amount of sodium pentobarbital. After excising the hearts of theanimals, the infarcted regions of myocardial tissue were determined bystaining with triphenyl tetrazolium chloride (TTC). Those regions thatwere not stained indicated infarcted sites. On the other hand, ischemicregions were determined by injecting Evan's blue stain (4%, SigmaChemical Corp., St. Louis, Mo., USA) into the aortic artery at 80 mmHg.Tissue that has not become ischemic is stained blue by this stain, whileischemic regions appear white since this stain is not transported bycapillaries.

The left ventricle was severed to obtain atrioventricular rings and atotal of seven tissue sections were obtained. After weighing eachtissue, the tissue sections were incubated in 1% TTC solution at 37° C.to visualize infarcted regions and capture images thereof (refer toFishbein, et al., Am. Heart J., 101, 593-600, 1981). The results areshown in FIG. 1. In the figure, the left panel depicts anatrioventricular ring administered physiological saline for use as acontrol, while the right panel depicts an atrioventricular ringfollowing transplantation of Muse cells. The regions in these tissuesections that are surrounded by broken lines (constituting a portion ofcardiac muscle and papillary muscle) indicate infarcted regions thatwere not stained by TTC. Although infarcted regions similar to thosepresent in the control were observed in the tissue section transplantedwith Muse cells, those regions can be seen to be much narrower incomparison with the control.

Moreover, infarct size was calculated as the ratio (%) of infarctedregions to ischemic regions (FIG. 2).

Although infarct size was 30.4% in a control group (n=3), infarct sizefollowing transplantation of Muse cells (n=4) was 18.2%, therebydemonstrating that infarct size was significantly reduced by Muse cells.When this effect is calculated in terms of reduction rate, Muse cellswere determined to be able to reduce infarct size by about 40%.Moreover, a similar test was carried out after increasing the number ofspecimens (FIG. 8). The average value of infarct size in a physiologicalsaline control group (n=10) was 27.0%, that in an MSC cell (mesenchymalcell fraction) transplant group (n=10) was 21.0%, that in a Muse celltransplant group (n=10) was 13.9%, and that in a non-Muse cell (MSCcells not containing Muse cells) (n=4) was 22.8%. On the basis of theseresults as well, Muse cells were determined to have a considerableeffect on reduction of infarct size.

In addition, a histological examination was made of the reducing effecton infarct size by Masson's trichrome staining. After fixing heartsexcised in the manner described above in 10% formalin and embedding inparaffin, sections were prepared in the direction of the horizontalcross-section from each specimen so as to obtain atrioventricular rings.Subsequently, the atrioventricular rings were stained with Masson'strichrome (MT) stain in accordance with ordinary methods to visualizeinfarcted regions of cardiac muscle (FIGS. 3 and 9). In the case of MTstaining, tissue composed of viable cells is stained red, while tissuein which collagen fibrosis has progressed is pale and appears to havelost color. Tissue exhibiting collagen fibrosis that is not stained byMT (infarcted sites) covers a wide area in a control group administeredphysiological saline. On the other hand, in left ventricular tissue of arabbit group transplanted with Muse cells, the pale areas were smallerthan in the control group, demonstrating that infarct size hasdecreased. In addition, papillary muscle was also characteristicallyobserved to have recovered from infarction. On the other hand, when leftventricle tissue of rabbits transplanted with MSC cells serving as acomparative control was compared with a control group administeredphysiological saline, although a reduction in infarct size was observed,the reducing effect on infarct size was less potent in comparison withthat of the Muse cell transplant group. In addition, recovery ofpapillary muscle observed in the Muse cell transplant group was notobserved in tissue transplanted with MSC cells.

Example 3: Differentiation of Muse Cells in Cardiac Tissue

A study was made as to whether or not the reduction of infarct sizeattributable to Muse cells observed in Example 2 was the result ofdifferentiation by Muse cells into myocardial cells. First, Muse cells(5×10⁵), inserted with a gene so as to express green fluorescent protein(GFP), were injected into a rabbit model of myocardial infarctionthrough an ear vein. Tissue sections were prepared in the same manner asExample 2 and the tissue was observed using fluorescent dyes for eachtype of tissue stain (FIG. 4). As is shown in the left panel of FIG. 4,the white broken line indicates a boundary line between an infarctedportion and a non-infarcted portion, with the portion above and to theright of the boundary line indicating infarcted sites and the portionbelow and to the left of the boundary line indicating non-infarctedsites. The center panel depicts the results of staining myocardial cellsred with rhodamine-phalloidin stain in accordance with ordinary methods.This staining enables infarcted sites and non-infarcted sites to beclearly distinguished. In addition, the right panel indicates an imageobtained from GFP staining, and Muse cells introduced with GFP gene(green) are indicated as having selectively accumulated at infractedsites. An image obtained by superimposing these two images is shown inthe left panel. As a result, a large number of cells where red and greencolors overlap are present at the infarcted sites, thereby suggestingthat transplanted Muse cells differentiated into myocardial cells.

Moreover, in order to investigate the differentiated state ofGFP-positive Muse cells that integrated into infarcted sites, thesecells were examined for the presence or absence of expression of atrialnatriuretic peptide (ANP) in accordance with ordinary methods. This ANPis known to be expressed in juvenile myocardial cells. In FIG. 5, thegreen color indicates GFP stain (Muse cells), the red color indicatesANP stain and the blue color indicates DAPI stain (which is used tostain cell nuclei). As can be understood from the left panel of FIG. 5,since these three types of fluorescence were observed within a pluralityof the same cells, transplanted Muse cells were suggested to includecells that were differentiating into myocardial cells.

Next, an examination was made of the presence or absence of theexpression of troponin I, which is known to be a myocardial marker, inGFP-positive Muse cells that integrated into infarcted sites. Introponin I staining, mouse anti-human troponin I antibody (ChemicalInternational, Inc.), which cross-reacts with rabbit troponin I, wasused as primary antibody. In FIG. 10, the green color indicates GFPstain (Muse cells), the red color indicates troponin I stain and theblue color indicates DAPI stain (which is used to stain cell nuclei). Ascan be understood from the right panel in FIG. 10, since these threetypes of fluorescence were observed within a plurality of the samecells, transplanted Muse cells were suggested to include cells that weredifferentiating into myocardial cells in the same manner as demonstratedby the aforementioned results (FIG. 5).

Moreover, an examination was made of the presence or absence ofexpression of CD31, which is known to be a vascular endothelial cellmarker, in order to investigate the differentiated state of Muse cellsthat integrated into infarcted sites. More specifically, mouseanti-human CD31 monoclonal antibody (acquired from Dako Corp.), whichcross-reacts with rabbit vascular endothelial cells, was used as primaryantibody, and cells that expressed CD31 were histochemically stained. Asis clear from the micrographs shown in FIG. 13, CD31-positivemicrovascular density at infarcted sites was higher in tissuetransplanted with Muse cells than in the other transplant groups. On thebasis thereof, the possibility was suggested that the transplanted Musecells differentiate into vascular endothelial cells at infarcted sites.

Example 4: Evaluation of Improvement of Cardiac Function byTransplantation of Muse Cells

An examination was made of cardiac function following transplantation ofMuse cells by using changes in blood pressure over time as an indicator(±dp/dt, p: blood pressure, t: time), and was assessed on the basis ofimages of cross-sections of the left ventricle obtained by 2Dechocardiography. In these experiments, rabbits administeredphysiological saline 24 hours after reperfusion (control group, n=3) anda group of rabbits administered Muse cells (n=4) were used. First,measurement of changes in blood pressure over time was carried out bymildly anesthetizing each rabbit 14 days after reperfusion with 10 mg/kgof sodium pentobarbital, and inserting a catheter equipped with amicromanometer (SRP 407, Millar Instruments Inc.) into the leftventricle of the rabbits through the carotid artery. The values of+dp/dt, which represents cardiac systolic function of the leftventricle, and −dp/dt, which represents cardiac diastolic function ofthe left ventricle, that were obtained with this catheter were recorded.The results are shown in FIG. 6. Both cardiac systolic function (+dp/dt)(top of FIG. 6) and cardiac diastolic function (−dp/dt) (bottom of FIG.6) demonstrated significant improvement of cardiac function in a grouptransplanted with Muse cells in comparison with a control group.

Moreover, the results of having examined cardiac function using changesin blood pressure over time in the same manner as previously describedafter increasing the number of specimens are shown in FIG. 11. Bothcardiac systolic function (+dp/dt) (top of FIG. 11) and cardiacdiastolic function (−dp/dt) (bottom of FIG. 11) demonstrated significantimprovement of cardiac function in a rabbit group transplanted with Musecells (n=10) in comparison with a control group (n=10), an MSC celltransplant group (n=10) and a non-Muse cell transplant group (n=9).

Next, 2D echocardiography was carried out in order to further confirmcardiac function in the aforementioned rabbits (control group and Musecell transplant group). In this 2D echocardiography, images of thehearts of the rabbits were captured using an ultrasound diagnosticimaging system for use with animals (SSD2000, Aloka Corp.). Parasternallong axis cross-sections of the left ventricle obtained by measuring areshown in FIG. 7. The left panel indicates an image of the left ventricleof a control rabbit, while the right panel indicates an image of theleft ventricle of a rabbit transplanted with Muse cells. The leftventricular end-diastolic dimension (LVDd) in the control was 22.2 mm.In contrast, since the value in the rabbit transplanted with Muse cellswas smaller at 19.5 mm, infract size can be understood to have beenreduced by Muse cells. Moreover, measurement of ejection fraction (EF),which is also used as an indicator of cardiac systolic function, yieldeda value of 34.1% in the control, while the value in the rabbittransplanted with Muse cells was 60.9%. This ejection fraction isrepresented as the ratio of a single cardiac output of the leftventricle to left ventricular end-diastolic volume. Normally, anejection volume of 55% or higher is considered to be normal in humans.Thus, the aforementioned measurement result suggests that cardiacfunction was returned to normal by transplantation of Muse cells inrabbits in comparison with the control. In addition, measurement of leftventricular fractional shortening (FS), which is also used as anindicator of cardiac systolic function in the same manner as EF, yieldeda value of 15.1% in the control and value of 30.4% in the rabbittransplanted with Muse cells. This left ventricular fractionalshortening is represented as a percentage by measuring left ventricularend-diastolic dimension and left ventricular end-systolic dimensionusing M-mode echocardiograms obtained by imaging, and dividing thedifference thereof by the left ventricular end-diastolic dimension.Normally, the normal value in humans is considered to be within therange of 30% to 50%. Thus, the aforementioned measurement resultssuggest that cardiac function was returned to normal by transplantationof Muse cells in rabbits in comparison with the control.

In order to reconfirm the restoration of cardiac function to normal inrabbits following transplantation of Muse cells, LVDd, EF, FS (aspreviously described) and left ventricular end-systolic dimension (LVDs)were measured for control rabbits, rabbits transplanted with MSC cellsand rabbits transplanted with non-Muse cells. Similar to theaforementioned results, LVDd, EF and FS all demonstrated that cardiacfunction was returned to normal in rabbits as a result of transplantingMuse cells. In addition, with respect to LVDs as well, in contrast toLVDs being an average of 18.3 mm in the control rabbits, the averagevalue in rabbits transplanted with Muse cells was smaller at 13.8 mm,thereby suggesting that cardiac function was returned to normal as aresult of transplanting Muse cells.

INDUSTRIAL APPLICABILITY

The cell preparation of the present invention is able to regeneratecardiac muscle at an infarcted site, reduce infarct size and improvecardiac function by administering transvenously into a cardiacinfarction model, and can be applied to the treatment of myocardialinfarction, and particularly serious massive myocardial infarction andheart failure associated therewith in humans.

All publications and patent documents cited in the present descriptionare incorporated throughout the description by reference. Furthermore,although specific embodiments of the present invention have beenexplained in the present description for the purpose of exemplification,it can be easily understood by a person with ordinary skill in the artthat the present invention may be modified in various ways withoutdeparting from the spirit and scope thereof.

The invention claimed is:
 1. A method of treating myocardial infarctionin a subject in need thereof, the method comprising: administering tosaid subject a cell preparation comprising isolated pluripotent stemcells positive for SSEA-3, wherein the pluripotent stem cells areisolated from a mesenchymal cell population obtained from mesenchymaltissue or cultured mesenchymal cells, wherein said pluripotent stemcells are isolated by subjecting said mesenchymal cell population toexternal stress stimulation or cell sorting, to thereby treat themyocardial infarction, and wherein the pluripotent stem cells have aplurality of properties comprising: (i) CD105-positivity; (ii) low orabsent telomerase activity; (iii) ability to differentiated intoembryonic endoderm, ectoderm, and mesoderm germ layers; (iv) absence ofneoplastic proliferation; and (v) ability to self-renew.
 2. The methodaccording to claim 1, wherein the cell preparation contains a cellfraction wherein the pluripotent stem cells positive for SSEA-3 havebeen concentrated by external stress stimulation.
 3. The methodaccording to claim 1 for treatment of heart failure following seriousmassive myocardial infarction in a human.
 4. The method according toclaim 1, wherein the pluripotent stem cells are CD117-negative andCD146-negative.
 5. The method according to claim 1, wherein thepluripotent stem cells are CD117-negative, CD146-negative, NG2-negative,CD34-negative, vWF-negative and CD271-negative.
 6. The method accordingto claim 1, wherein the pluripotent stem cells are CD34-negative,CD117-negative, CD146-negative, CD271-negative, NG2-negative,vWF-negative, Sox10-negative, Snail-negative, Slug-negative,Tyrp1-negative and Dct-negative.
 7. The method according to claim 1,wherein the pluripotent stem cells have the ability to integrate intothe site of myocardial infarction.
 8. The method according to claim 1,wherein the pluripotent stem cells have the ability to differentiateinto myocardial cells.
 9. The method according to claim 1, wherein thepluripotent stem cells have the ability to differentiate into vascularendothelial cells.
 10. The method according to claim 1, wherein thepluripotent stem cells are administered into a vein or coronary arteryof a subject within 1 month after ischemia one to ten times in atherapeutically effective amount of 1×10³ cells/individual to 1×10⁶cells/individual.
 11. The method according to claim 1, wherein at leastone cardiac function indicator, selected from the group consisting ofchange in left ventricular pressure over time, left ventricularend-diastolic dimension (LVDd), ejection fraction (EF), left ventricularfractional shortening (FS) and left ventricular end-systolic dimension(LVDs), is restored to the normal values.
 12. The method according toclaim 2, wherein the external stress stimulation is a member selectedfrom a group consisting of protease treatment, culturing underoxygen-deficient conditions, culturing under phosphate-deficientconditions, culturing under serum-deficient conditions, culturing underpoor nutritional conditions, culturing under exposure to heat shock,culturing under mechanical stimulation, culturing under shakingtreatment, culturing under pressure treatment and physical shock, and acombination of a plurality thereof.
 13. The method according to claim 1,wherein the cell preparation is administered into a vein or coronaryartery of said subject within 1 month after ischemia in atherapeutically effective amount of 1.7×10⁵ cells/kg to 2.5×10⁵ cells/kgper individual mammal based on body weight.
 14. The method according toclaim 1, wherein the route of administration is intravenous.